Peroxisomes are single membrane-bound organelles that function to compartmentalize certain metabolic reactions critical to human and plant development. I am studying peroxisomal processes in the model plant Arabidopsis thaliana, with a focus the import of matrix proteins from the cytoplasm into the organelle matrix. This import depends on more than a dozen peroxin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing a peroxisome targeting sequence (PTS) into the organelle. PEX5 is the PTS1 receptor, PEX7 is the PTS2 receptor, and in both plants and mammals, PEX7 depends upon PEX5 binding to deliver PTS2 cargo into the peroxisome. I propose to elucidate the functions of Arabidopsis PEX7 in peroxisomal matrix protein import. I will characterize pex7 mutants isolated through forward and reverse genetic screens in physiological and biochemical assays. I will examine localization of various peroxisomally targeted GFP derivatives and endogenous peroxisomal enzymes in the pex7 mutants, and determine whether the pex7 mutations disrupt PEX7-cargo binding, PEX7-PEX5 interactions, or PEX7 stability. I will characterize the interactions between mutant and wild-type PEX7 and PEX5 derivatives using the yeast two-hybrid assay, overexpression studies, and double mutant analyses. Finally, I will explore an alternate means for targeting PEX7 for PEX5 binding. In humans, deficiencies in PEX7 and other peroxins underlie the peroxisomal biogenesis disorders, which are frequently lethal in early infancy. Successful completion of these experiments will advance our understanding of peroxisome biogenesis and metabolism in a genetically distinct model system, which will allow the continued refinement of our understanding of these essential organelles.